Ever increasing attention is being paid to detection and analysis of low concentrations of analytes in various biologic and organic environments. Qualitative analysis of such analytes is generally limited to the higher concentration levels, whereas quantitative analysis usually requires labeling with a radioisotope or fluorescent reagent. Such procedures are generally time consuming and inconvenient.
Solid-state sensors and particularly biosensors have received considerable attention lately due to their increasing utility in chemical, biological, and pharmaceutical research as well as disease diagnostics. In general, biosensors consist of two components: a highly specific recognition element and a transducing structure that converts the molecular recognition event into a quantifiable signal. Biosensors have been developed to detect a variety of biomolecular complexes including oligonucleotide pairs, antibody-antigen, hormone-receptor, enzyme-substrate and lectin-glycoprotein interactions. Signal transductions are generally accomplished with electrochemical field-effect transistor, optical absorption, fluorescence or interferometric devices.
It is known that the intensity of the visible reflectivity changes of a porous silicon film can be utilized in a simple biological sensor, as disclosed in U.S. Pat. No. 6,248,539 to Ghadiri et al. As disclosed therein, the detection and measurement of wavelength shifts in the interference spectra of a porous semiconductor substrate, such as a silicon substrate, make possible detection, identification and quantification of small molecules. While such a biological sensor is certainly useful, detection of a reflectivity shift is complicated by the presence of a broad peak rather than one or more sharply defined luminescent peaks.
Various sensors that include a porous multilayer semiconductor structure having layers of alternating porosity have been developed. Upon binding of an analyte molecule, a detectable change occurs in a refractive index of the semiconductor structure to signal binding of the sensor to the analyte. The central layer of such a multilayered structure positioned between upper and lower layers (Bragg reflectors), forms a microcavity resonator. This microcavity resonator confines the luminescence generated in the central layer of the microcavity by the two layers that act as Bragg reflectors so that the photoluminescence spectrum is composed of multiple sharp and narrow peaks with Full-Width Half-Maximum, or FWHM, values of about 3 nm (Chan et al., Phys. Stat. Sol. A 182:541-546 (2000). Upon a refractive index change, the photoluminescent spikes shift, thereby generating a detectable differential signal. However, the binding event provides only approximate information regarding the nature of the molecule that binds.
Raman spectroscopy or surface plasmon resonance has also been used seeking to achieve the goal of sensitive and accurate detection or identification of individual molecules from biological samples. When light passes through a medium of interest, a certain amount of the light becomes diverted from its original direction in a phenomenon known as scattering. Some of the scattered light also differs in frequency from the original excitatory light, due to the absorption of light and excitation of electrons to a higher energy state, followed by light emission at a different wavelength. The difference of the energy of the absorbed light and the energy of the emitted light matches the vibrational energy of the medium. This phenomenon is known as Raman scattering, and the method to characterize and analyze the medium or molecule of interest with the Raman scattered light is called Raman spectroscopy. The wavelengths of the Raman emission spectrum are characteristic of the chemical composition and structure of the Raman scattering molecules in a sample, while the intensity of Raman scattered light is dependent on the concentration of molecules in the sample.
It has been observed that molecules near roughened silver surfaces show enhanced Raman scattering of as much as two orders of magnitude or more. This surface enhanced Raman scattering (SERS) effect is related to the phenomenon of plasmon resonance, wherein metal nanoparticles or metal coatings exhibit a pronounced optical resonance in response to incident electromagnetic radiation, due to the collective coupling of conduction electrons in the metal. In essence, nanoparticles of gold, silver, copper and certain other metals can function to enhance the localized effects of electromagnetic radiation. Molecules located in the vicinity of such particles exhibit a much greater sensitivity for Raman spectroscopic analysis. SERS is the technique to utilize this surface enhanced Raman scattering effect to characterize and analyze biological molecules of interest.
Sodium chloride and lithium chloride have been identified as chemicals that enhance the SERS signal when applied to a metal nanoparticle or metal coated surface before or after the molecule of interest has been introduced. However, the technique of using these chemical enhancers has not proved sensitive enough to reliably detect low concentrations of analyte molecules, such as single nucleotides or proteins. Only one type of nucleotide, deoxyadenosine monophosphate, and only one type of protein, hemoglobin, have been detected at single molecule level. As a result SERS has not been viewed as suitable for analyzing the protein content of a complex biological sample, such as blood plasma.
Thus a need exists in the art for a method of biomolecule detection that provides information regarding the characteristics of the bound molecule and for reliably detecting and/or identifying individual molecules using a Raman spectroscopic analytical technique. In addition, there is also a need in the art for quick and simple means of qualitatively and quantitatively detecting biomolecules at low concentration levels.